Priming of Colorectal Tumor-Associated Fibroblasts with Zoledronic Acid Conjugated to the Anti-Epidermal Growth Factor Receptor Antibody Cetuximab Elicits Anti-Tumor Vδ2 T Lymphocytes.

Tumor-associated fibroblasts (TAF) exert immunosuppressive effects in colorectal carcinoma (CRC), impairing the recognition of tumor cells by effector lymphocytes, including Vδ2 T cells. Herein, we show that CRC-derived TAF can be turned by zoledronic acid (ZA), in soluble form or as antibody-drug conjugate (ADC), into efficient stimulators of Vδ2 T cells. CRC-TAF, obtained from patients, express the epidermal growth factor receptor (EGFR) and the butyrophilin family members BTN3A1/BTN2A1. These butyrophilins mediate the presentation of the phosphoantigens, accumulated in the cells due to ZA effect, to Vδ2 T cells. CRC-TAF exposed to soluble ZA acquired the ability to trigger the proliferation of Vδ2 T cells, in part represented by effector memory cells lacking CD45RA and CD27. In turn, expanded Vδ2 T cells exerted relevant cytotoxic activity towards CRC cells and CRC-TAF when primed with soluble ZA. Of note, also the ADC made of the anti-EGFR cetuximab (Cet) and ZA (Cet-ZA), that we recently described, induced the proliferation of anti-tumor Vδ2 T lymphocytes and their activation against CRC-TAF. These findings indicate that ZA can educate TAF to stimulate effector memory Vδ2 T cells; the Cet-ZA ADC formulation can lead to the precise delivery of ZA to EGFR+ cells, with a double targeting of TAF and tumor cells.

SB202190 Predicts BRAF-Activating Mutations in Primary Colorectal Cancer Organoids via Erk1-2 Modulation.

The p38 inhibitor SB202190 is a necessary component of the medium used for normal colorectal mucosa cultures. Sato et al. suggested that the primary activity of SB202190 may be EGFR signaling stabilization, causing an increased phosphorylation of Erk1-2 sustaining organoid proliferation. However, the growth of some colorectal cancer (CRC)-derived organoid cultures is inhibited by this molecule via an unknown mechanism. We biochemically investigated SB202190 activity on a collection of 25 primary human CRC organoids, evaluating EGFR, Akt and Erk1-2 activation using Western blot. We found that Erk1-2 phosphorylation was induced by SB202190 in 20 organoid cultures and inhibited in 5 organoid cultures. A next-generation sequencing (NGS) analysis revealed that the inhibition of p-Erk1-2 signaling corresponded to the cultures with BRAF mutations (with four different hits, one being undescribed), while p-Erk1-2 induction was apparently unrelated to other mutations involving the EGFR pathway (Her2, KRAS and NRAS). We found that SB202190 mirrored the biochemical activity of the BRAF inhibitor Dabrafenib, known to induce the paradoxical activation of p-Erk1-2 signaling in BRAF wild-type cells. SB202190 was a more effective inhibitor of BRAF-mutated organoid growth in the long term than the specific BRAF inhibitors Dabrafenib and PLX8394. Overall, SB202190 can predict BRAF-activating mutations in patient-derived organoids, as well as allowing for the identification of new BRAF variants, preceding and enforcing NGS data.

Targeting of colorectal cancer organoids with zoledronic acid conjugated to the anti-EGFR antibody cetuximab.

BACKGROUND: Antibody-drug conjugates (ADC) are essential therapeutic options to treat solid and hematological cancers. The anti-epidermal growth factor-receptor (EGFR) antibody cetuximab (Cet) is used for the therapy of colorectal carcinoma (CRC). Anti-CRC Vδ2 cytolytic T lymphocytes can be elicited by the priming of tumor cells with the aminobisphosphonate zoledronic acid (ZA) and consequent presentation of isopentenyl pyrophosphates through butyrophilin (BTN) family members such as BTN3A1 and BTN2A1. A major drawback that impairs the targeting of ZA to CRC is the bone tropism of aminobisphosphonates. METHODS: The phosphoric group of ZA was linked to free amino groups of Cet in the presence of imidazole following the labeling of phosphoric groups of DNA to amino groups of proteins. The generation of Cet-ZA ADC was confirmed by matrix assisted laser desorption ionization mass spectrometry and inductively coupled plasma-mass spectrometry analysis. Thirteen CRC organoids were obtained with a chemically defined serum-free medium in Geltrex domes. Proliferation and activation of cytolytic activity against CRC organoids by Vδ2 T cells was detected with flow cytometry, crystal violet and cytotoxic probe assays and image analysis. Immunohistochemistry and quantification of BTN3A1 or BTN2A1 expression and the number of tumor infiltrating Vδ2 T cells in CRC were performed by automatic immunostaining, whole slide scanning and computerized analysis of digital pathology imaging. RESULTS: The novel ADC Cet-ZA was generated with a drug antibody ratio of 4.3 and displayed a reactivity similar to the unconjugated antibody. More importantly, patient-derived CRC organoids, or CRC tumor cell suspensions, could trigger the expansion of Vδ2 T cells from peripheral blood and tumor infiltrating lymphocytes when primed with Cet-ZA. Furthermore, Cet-ZA triggered Vδ2 T cell-mediated killing of CRC organoids. The expression of BTN3A1 and BTN2A1 was detected not only in CRC organoids but also in CRC specimens, together with a considerable amount of tumor infiltrating Vδ2 T cells. CONCLUSIONS: These findings are proof of concept that the Cet-ZA ADC can be used to target specifically CRC organoids and may suggest a new experimental approach to deliver aminobisphosphonates to EGFR+ solid tumors.

Analysis of A Disintegrin and Metalloprotease 17 (ADAM17) Expression as a Prognostic Marker in Ovarian Cancer Patients Undergoing First-Line Treatment Plus Bevacizumab.

To find prognostic factors for advanced ovarian cancer patients undergoing first-line therapy with carboplatin, paclitaxel and bevacizumab, we investigated the expression of a disintegrin and metalloprotease 17 (ADAM17) in cancer tissues. ADAM17 has been involved in ovarian cancer development, progression and cell resistance to cisplatin. Tissue microarrays from 309 ovarian cancer patients enrolled in the MITO16A/MANGO-OV2 clinical trial were analyzed by immunohistochemistry for ADAM17 protein expression. Intensity and extent of staining were combined into a semi-quantitative visual grading system (H score) which was related to clinicopathological characteristics of cases and the clinical outcome of patients by univariate and multivariate Cox regression models. ADAM17 immunostaining was detected in most samples, mainly localized in the tumor cells, with variable intensity across the cohort. Kaplan-Meier survival curves, generated according to the best cut-off value for the ADAM17 H score, showed that high ADAM17 expression was associated with worse prognosis for PFS and OS. However, after the application of a shrinkage procedure to adjust for overfitting hazard ratio estimates, the ADAM17 value as prognostic factor was lost. As subgroup analysis suggested that ADAM17 expression could be prognostically relevant in cases with no residual disease at baseline, further studies in this patient category may be worth planning.

Lysyl-Oxidase Dependent Extracellular Matrix Stiffness in Hodgkin Lymphomas: Mechanical and Topographical Evidence.

PURPOSE: The biochemical composition and architecture of the extracellular matrix (ECM) is known to condition development and invasiveness of neoplasms. To clarify this point, we analyzed ECM stiffness, collagen cross-linking and anisotropy in lymph nodes (LN) of Hodgkin lymphomas (HL), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL), compared with non-neoplastic LN (LDN). METHODS AND RESULTS: We found increased elastic (Young's) modulus in HL and advanced FL (grade 3A) over LDN, FL grade 1-2 and DLBCL. Digital imaging evidenced larger stromal areas in HL, where increased collagen cross-linking was found; in turn, architectural modifications were documented in FL3A by scanning electron microscopy and enhanced anisotropy by polarized light microscopy. Interestingly, HL expressed high levels of lysyl oxidase (LOX), an enzyme responsible for collagen cross-linking. Using gelatin scaffolds fabricated with a low elastic modulus, comparable to that of non-neoplastic tissues, we demonstrated that HL LN-derived mesenchymal stromal cells and HL cells increased the Young's modulus of the extracellular microenvironment through the expression of LOX. Indeed, LOX inhibition by ß-aminopropionitrile prevented the gelatin stiffness increase. CONCLUSIONS: These data indicate that different mechanical, topographical and/or architectural modifications of ECM are detectable in human lymphomas and are related to their histotype and grading.

Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Extension of Collagen Deposition in COVID-19 Post Mortem Lung Samples and Computed Tomography Analysis Findings.

Lung fibrosis has specific computed tomography (CT) findings and represents a common finding in advanced COVID-19 pneumonia whose reversibility has been poorly investigated. The aim of this study was to quantify the extension of collagen deposition and aeration in postmortem cryobiopsies of critically ill COVID-19 patients and to describe the correlations with qualitative and quantitative analyses of lung CT. Postmortem transbronchial cryobiopsy samples were obtained, formalin fixed, paraffin embedded and stained with Sirius red to quantify collagen deposition, defining fibrotic samples as those with collagen deposition above 10%. Lung CT images were analyzed qualitatively with a radiographic score and quantitatively with computer-based analysis at the lobe level. Thirty samples from 10 patients with COVID-19 pneumonia deceased during invasive mechanical ventilation were included in this study. The median [interquartile range] percent collagen extension was 6.8% (4.6-16.2%). In fibrotic compared to nonfibrotic samples, the qualitative score was higher (260 (250-290) vs. 190 (120-270), p = 0.036) while the gas fraction was lower (0.46 (0.32-0.47) vs. 0.59 (0.37-0.68), p = 0.047). A radiographic score above 230 had 100% sensitivity (95% confidence interval, CI: 66.4% to 100%) and 66.7% specificity (95% CI: 41.0% to 92.3%) to detect fibrotic samples, while a gas fraction below 0.57 had 100% sensitivity (95% CI: 66.4% to 100%) and 57.1% specificity (95% CI: 26.3% to 88.0%). In COVID-19 pneumonia, qualitative and quantitative analyses of lung CT images have high sensitivity but moderate to low specificity to detect histopathological fibrosis. Pseudofibrotic CT findings do not always correspond to increased collagen deposition.

Three-Dimensional Culture Models to Study Innate Anti-Tumor Immune Response: Advantages and Disadvantages.

Several approaches have shown that the immune response against tumors strongly affects patients' clinical outcome. Thus, the study of anti-tumor immunity is critical to understand and potentiate the mechanisms underlying the elimination of tumor cells. Natural killer (NK) cells are members of innate immunity and represent powerful anti-tumor effectors, able to eliminate tumor cells without a previous sensitization. Thus, the study of their involvement in anti-tumor responses is critical for clinical translation. This analysis has been performed in vitro, co-incubating NK with tumor cells and quantifying the cytotoxic activity of NK cells. In vivo confirmation has been applied to overcome the limits of in vitro testing, however, the innate immunity of mice and humans is different, leading to discrepancies. Different activating receptors on NK cells and counter-ligands on tumor cells are involved in the antitumor response, and innate immunity is strictly dependent on the specific microenvironment where it takes place. Thus, three-dimensional (3D) culture systems, where NK and tumor cells can interact in a tissue-like architecture, have been created. For example, tumor cell spheroids and primary organoids derived from several tumor types, have been used so far to analyze innate immune response, replacing animal models. Herein, we briefly introduce NK cells and analyze and discuss in detail the properties of 3D tumor culture systems and their use for the study of tumor cell interactions with NK cells.

MCM2 and Carbonic Anhydrase 9 Are Novel Potential Targets for Neuroblastoma Pharmacological Treatment.

To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.

Aspartate ß-hydroxylase targeting in castration-resistant prostate cancer modulates the NOTCH/HIF1α/GSK3ß crosstalk.

Castration-resistant prostate cancer (CRPC) is an incurable stage of the disease. A multivariate principal component analysis on CRPC in vitro models identified aspartyl (asparaginyl) ß hydrolase (ASPH) as the most relevant molecule associated with the CRPC phenotype. ASPH is overexpressed in various malignant neoplasms and catalyzes the hydroxylation of aspartyl and asparaginyl residues in the epidermal growth factor (EGF)-like domains of proteins like NOTCH receptors and ligands, enhancing cell motility, invasion and metastatic spread. Bioinformatics analyses of ASPH in prostate cancer (PCa) and CRPC datasets indicate that ASPH gene alterations have prognostic value both in PCa and CRPC patients. In CRPC cells, inhibition of ASPH expression obtained through specific small interfering RNA or culturing cells in hypoxic conditions, reduced cell proliferation, invasion and cyclin D1 expression through modulation of the NOTCH signaling. ASPH and HIF1α crosstalk, within a hydroxylation-regulated signaling pathway, might be transiently driven by the oxidative stress evidenced inside CRPC cells. In addition, increased phosphorylation of GSK3ß by ASPH silencing demonstrates that ASPH regulates GSK3ß activity inhibiting its interactions with upstream kinases. These findings demonstrate the critical involvement of ASPH in CRPC development and may represent an attractive molecular target for therapy.

Erratum: Benelli, R., et al. Aspartate-ß-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer. Cancers 2020, 12, 971.

The authors wish to make the following corrections to this paper [...].

Aspartate-ß-Hydroxylase: A Promising Target to Limit the Local Invasiveness of Colorectal Cancer.

Colorectal cancer's (CRC) ability to invade local tissues and lymph nodes and generate distant metastases is the key for TNM classification. Aspartate-ß-hydroxylase (ASPH), a transmembrane protein that catalyzes Notch receptors and ligand activation, is involved in tumor invasion. Because Notch is involved in gut homeostasis, it could be a target for CRC therapy. ASPH mRNA and protein expression, promoter methylation and gene copy numbers were evaluated using the TCGA and CPTAC human CRC datasets. Using digital pathology, ASPH was scored in the luminal area (LM), center tumor (CT) and invasive margin (IM) of 100 human CRCs. The effect of ASPH targeting on invasiveness and viability was tested by siRNA knockdown and small molecule inhibitors (SMI). Bioinformatics analysis showed increased expression of ASPH mRNA and protein in CRC, paired with a decreased methylation profile. ASPH genetic gain or amplification was frequent (56%), while deletion was rare (0.03%). Digital pathology analysis showed that ASPH exerted its pathological activity in the invasive margin of the tumor, affecting invasive front morphology, tumor budding and patients' overall survival. In vitro, ASPH targeting by siRNA or SMI reduced cell invasion and growth and caused Notch-1 downregulation. This study demonstrates that ASPH targeting by specific inhibitors could improve CRC treatment strategies.

Evaluation of Glycosylated PTGS2 in Colorectal Cancer for NSAIDS-Based Adjuvant Therapy.

Observational/retrospective studies indicate that prostaglandin-endoperoxide synthase-2 (PTGS2) inhibitors could positively affect colorectal cancer (CRC) patients' survival after diagnosis. To obtain an acceptable cost/benefit balance, the inclusion of PTGS2 inhibitors in the adjuvant setting needs a selective criterion. We quantified the 72 kDa, CRC-associated, glycosylated form of PTGS2 in 100 frozen CRC specimens and evaluated PTGS2 localization by IHC in the same tumors, scoring tumor epithelial-derived and stroma-derived fractions. We also investigated the involvement of interleukin-1 beta (IL1ß) in PTGS2 induction, both in vitro and in CRC lysates. Finally, we used overall survival (OS) as a criterion for patient selection. Glycosylated PTGS2 can be quantified with high sensibility in tissue lysates, but the expression in both tumor and stromal cells limits its use for predictive purposes. Immunohistochemistry (IHC) analysis indicates that stromal PTGS2 expression could exert a protective role on patient OS. Stromal PTGS2 was prevalently expressed by cancer-associated fibroblasts exerting a barrier function near the gut lumen, and it apparently favored the antitumor M1 macrophage population. IL1ß was directly linked to gPTGS2 expression both in vitro and in tumors, but its activity was apparently prevalent on the stromal cell population. We suggest that stromal PTGS2 could exert a positive effect on patients OS when expressed in the luminal area of the tumor.

Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis.

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies.

Multifocal Signal Modulation Therapy by Celecoxib: A Strategy for Managing Castration-Resistant Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. METHODS: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. RESULTS: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-κB activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. CONCLUSIONS: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

Human Gut-Associated Natural Killer Cells in Health and Disease.

It is well established that natural killer (NK) cells are involved in both innate and adaptive immunity. Indeed, they can recognize molecules induced at the cell surface by stress signals and virus infections. The functions of NK cells in the gut are much more complex. Gut NK cells are not precisely organized in lymphoid aggregates but rather scattered in the epithelium or in the stroma, where they come in contact with a multitude of antigens derived from commensal or pathogenic microorganisms in addition to components of microbiota. Furthermore, NK cells in the bowel interact with several cell types, including epithelial cells, fibroblasts, macrophages, dendritic cells, and T lymphocytes, and contribute to the maintenance of immune homeostasis and development of efficient immune responses. NK cells have a key role in the response to intestinal bacterial infections, primarily through production of IFNγ, which can stimulate recruitment of additional NK cells from peripheral blood leading to amplification of the anti-bacterial immune response. Additionally, NK cells can have a role in the pathogenesis of gut autoimmune inflammatory bowel diseases (IBDs), such as Crohn's Disease and Ulcerative Colitis. These diseases are considered relevant to the generation of gastrointestinal malignancies. Indeed, the role of gut-associated NK cells in the immune response to bowel cancers is known. Thus, in the gut immune system, NK cells play a dual role, participating in both physiological and pathogenic processes. In this review, we will analyze the known functions of NK cells in the gut mucosa both in health and disease, focusing on the cross-talk among bowel microenvironment, epithelial barrier integrity, microbiota, and NK cells.

Anthropometric and glucometabolic changes in an aged mouse model of lipocalin-2 overexpression.

BACKGROUND: Lipocalin-2 (LCN2) is widely expressed in the organism with pleiotropic roles. In particular, its overexpression correlates with tissue stress conditions including inflammation, metabolic disorders, chronic diseases and cancer. OBJECTIVES: To assess the effects of systemic LCN2 overexpression on adipose tissue and glucose metabolism. SUBJECTS: Eighteen-month-old transgenic mice with systemic LCN2 overexpression (LCN2-Tg) and age/sex-matched wild-type mice. METHODS: Metabolic cages; histology and real-time PCR analysis; glucose and insulin tolerance tests; ELISA; flow cytometry; microPET and serum analysis. RESULTS: LCN2-Tg mice were smaller compared to controls but they ate (P = 0.0156) and drank (P = 0.0057) more and displayed a higher amount of visceral adipose tissue. Furthermore, LCN2-Tg mice with body weight ≥20 g showed adipocytes with a higher cell area (P < 0.0001) and altered expression of genes involved in adipocyte differentiation and inflammation. In particular, mRNA levels of adipocyte-derived Pparg (P ≤ 0.0001), Srebf1 (P < 0.0001), Fabp4 (P = 0.056), Tnfa (P = 0.0391), Il6 (P = 0.0198), and Lep (P = 0.0003) were all increased. Furthermore, LCN2-Tg mice displayed a decreased amount of basal serum insulin (P = 0.0122) and a statistically significant impaired glucose tolerance and insulin sensitivity consistent with Slc2a2 mRNA (P ≤ 0.0001) downregulated expression. On the other hand, Insr mRNA (P ≤ 0.0001) was upregulated and correlated with microPET analysis that demonstrated a trend in reduced whole-body glucose consumption and MRGlu in the muscles and a significantly reduced MRGlu in brown adipose tissue (P = 0.0247). Nevertheless, an almost nine-fold acceleration of hexokinase activity was observed in the LCN2-Tg mice liver compared to controls (P = 0.0027). Moreover, AST and ALT were increased (P = 0.0421 and P = 0.0403, respectively), which indicated liver involvement also demonstrated by histological staining. CONCLUSIONS: We show that LCN2 profoundly impacts adipose tissue size and function and glucose metabolism, suggesting that LCN2 should be considered as a risk factor in ageing for metabolic disorders leading to obesity.

Targeting the Epidermal Growth Factor Receptor Can Counteract the Inhibition of Natural Killer Cell Function Exerted by Colorectal Tumor-Associated Fibroblasts.

Mesenchymal stromal cells (MSC) present in the tumor microenvironment [usually named tumor-associated fibroblasts (TAF)] can exert immunosuppressive effects on T and natural killer (NK) lymphocytes, favoring tumor immune escape. We have analyzed this mechanism in colorectal carcinoma (CRC) and found that co-culture of NK cells with TAF can prevent the IL-2-mediated NKG2D upregulation. This leads to the impairment of NKG2D-mediated recognition of CRC cells, sparing the NK cell activation through DNAM1 or FcγRIIIA (CD16). In situ, TAF express detectable levels of epidermal growth factor receptor (EGFR); thus, the therapeutic anti-EGFR humanized antibody cetuximab can trigger the antibody-dependent cellular cytotoxicity of TAF, through the engagement of FcγRIIIA on NK cells. Importantly, in the tumor, we found a lymphoid infiltrate containing NKp46+CD3- NK cells, enriched in CD16+ cells. This population, sorted and cultured with IL-2, could be triggered via CD16 and via NKG2D. Of note, ex vivo NKp46+CD3- cells were able to kill autologous TAF; in vivo, this might represent a control mechanism to reduce TAF-mediated regulatory effect on NK cell function. Altogether, these findings suggest that MSC from the neoplastic mucosa (TAF) of CRC patients can downregulate the immune cell recognition of CRC tumor cells. This immunosuppression can be relieved by the anti-EGFR antibody used in CRC immunotherapy.

Zoledronate can induce colorectal cancer microenvironment expressing BTN3A1 to stimulate effector γδ T cells with antitumor activity.

Amino-bis-phosphonates (N-BPs) such as zoledronate (Zol) have been used in anticancer clinical trials due to their ability to upregulate pyrophosphate accumulation promoting antitumor Vγ9Vδ2 T cells. The butyrophilin 3A (BTN3A, CD277) family, mainly the BTN3A1 isoform, has emerged as an important structure contributing to Vγ9Vδ2 T cells stimulation. It has been demonstrated that the B30.2 domain of BTN3A1 can bind phosphoantigens (PAg) and drive the activation of Vγ9Vδ2 T cells through conformational changes of the extracellular domains. Moreover, BTN3A1 binding to the cytoskeleton, and its consequent membrane stabilization, is crucial to stimulate the PAg-induced tumor cell reactivity by human Vγ9Vδ2 T cells. Aim of this study was to investigate the relevance of BTN3A1 in N-BPs-induced antitumor response in colorectal cancer (CRC) and the cell types involved in the tumor microenvironment. In this paper, we show that (i) CRC, exposed to Zol, stimulates the expansion of Vδ2 T lymphocytes with effector memory phenotype and antitumor cytotoxic activity, besides sensitizing cancer cells to γδ T cell-mediated cytotoxicity; (ii) this effect is partially related to BTN3A1 expression and in particular with its cellular re-distribution in the membrane and cytoskeleton-associated fraction; (iii) BTN3A1 is detected in CRC at the tumor site, both on epithelial cells and on tumor-associated fibroblasts (TAF), close to areas infiltrated by Vδ2 T lymphocytes; (iv) Zol is effective in stimulating antitumor effector Vδ2 T cells from ex-vivo CRC cell suspensions; and (v) both CRC cells and TAF can be primed by Zol to trigger Vδ2 T cells.

LCN2 overexpression in bone enhances the hematopoietic compartment via modulation of the bone marrow microenvironment.

Lipocalin-2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress, and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2-Tg). In the bone marrow (BM) of LCN2-Tg mice we observed an increased number of phenotypically long-term hematopoietic stem cells (LT-HSC) that also displayed a higher proliferation rate compared to wild-type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2-Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2-Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2-Tg animals compared to aged-matched wt. The findings of a combined increase in the BM of the LCN2-Tg mice of SDF-1, SCF, and TIMP-1 levels along with the reduction of both MMP-9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment. This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice.